MOD1–DNAENGINEERING

Fall2010

Day2

Mod1:

In this module, you will create a plasmid that will be used in an assay to measure homologous

recombination activity in mammalian cells.

Background & Significance:

“Homology-Directed Repair” for double strand breaks

You will need to understand this material in order to analyze your data.

Sunlight

Pollution Food

Oxidative Radicals

Nitric Oxide Cigarette Smoke

Radiation

Base Lesions Single Strand Breaks Double Strand Breaks

After damage, what might happen?

=

Double Stranded DNA

Sister Chromatid

Double Stranded DNA

Double Strand Break

Damage

Resect to form 3’ overhang

Find Homology

Form D-loop

Polymerize DNA using invading strand with 3’OH as a primer and the homologous donor DNA as a template

Branch Migration

Bring extended ends together

Single Strand Annealing

Fill in gaps

+

=

See SDSA Prototypic Model

& Replication Fork

Animations by Justin Lo

Sunlight

Pollution Food

Oxidative Radicals

Nitric Oxide Cigarette Smoke

Radiation

Base Lesions Single Strand Breaks Double Strand Breaks

After damage, what might happen?

A 8-oxo-G

G C

8-oxo-G C

OH

T A

GT -> AT

Does this damage matter?

•  What can the cell do? Apoptose Necrose Nothing Fix it

•  If the cell is not dividing?

•  If the cell is dividing?

Human 8oxoG DNA Glycosylase

Dangers of Bulky Damage •  Polymerase is unable to bypass lesions •  This leads to replication fork breakdown

5’ 3’

3’ 5’ Leading

Strand Replication Fork Breakdown

Homologous Recombination Videos

•  http://web.mit.edu/engelward-lab/animations/forkHR.html

Possible pathways to resolve replication forks stalled at interstrand crosslinks. The stalled replication fork is recognized and cleaved in the leading-strand template to generate a one-sided DSB (steps 1-3).

Introduction of a second incision on the other side of the ICL (step 4a) allows the lesion to flip out and to be bypassed by TLS (Trans lesion synthesis) (green line). The DSB is processed to form a 3'-OH ending single-stranded tail (step 5a) and to initiate DNA strand invasion (step 6a). The replication fork is restored (steps 7a) and the lesion is bypassed by TLS (green line).

The DSB can also initiate DNA strand invasion using the homolog as a template (step 4b). DNA is synthesized across the lesion region (step 5b), disengaged (step 6b) and reinvasion of the sister chromatid behind the lesion site can lead to restoration of the replication fork and tolerance of the lesion (7b; the step from D-loop to recovered fork are not drawn and equivalent to Figure 2A, steps 3a-4a-5a).

Li and Heyer. Cell Research (2008)

Sunlight

Pollution Food

Oxidative Radicals

Nitric Oxide Cigarette Smoke

Radiation

Base Lesions Single Strand Breaks Double Strand Breaks

After damage, what might happen?

NHEJ

BRCA2-/-

Misrepair & Toxicity

BRCA2 is critical for repair of broken forks

What’s bad about repairing fork breakdown with NHEJ?

Raw data from Grigorova et al., Cytogen. and Gen. Res. 104:333-340 (2004)

BRCA2 -/- Chromosomes

www.rctradiology.com

2 15 11 10 Normal Human Chromosomes

Misrepair

Broken Fork Repair

NHEJ HR

Your Assay for Homologous Recombination

Δ5

Δ3

+ lipofect 48 hours

A Plasmid-Based Assay for Homologous Recombination in Mammalian Cells

•  AbouttheexperimentsinMod1

– HowdoesDNAdamagecausemuta>ons?

– Howisrecombina>onusedtofixdoublestrandbreaks?

– Overviewoftheexperimentsyouwillbedoing

•  Restric9onEnzymes

–  Basicsrestric>onenzymes

•  An9cipa9ngPoten9alProblems&Pi@alls

– Whatcontrolsareneededandwhy?

Overview of the Experiments in Mod1

Where you are, and where you are going

Restriction Enzymes

•  Where they come from? – Bacteria

•  What do they do? – Chop up the DNA of infecting phages

•  How do cells protect themselves? •  How can we use them?

Image from: Rosenberg, J. M. Curr. Opin. Struct. Biol. 1: 104-110 (1991)

5' - G A A T T C - 3' 3' - C T T A A G - 5'

EcoRI

GC AT

AT TA

TA CG

5' - G A A T T C - 3' 3' - C T T A A G - 5'

EcoRI

5' - G A A T T C - 3' 3' - C T T A A G - 5'

How do bugs keep from chopping themselves up?

M.HaeIII

Figure from R. Roberts, Annual Review of Biochemistry (1998) 67: 181-198.

5’-GGCC-3’ 3’-CCGG-5’

C

5MeC

“Cognate Methyltransferases”

Who’s Who?

Adenine

Guanine

Thymine

Cytosine

CH3

G:C

A:T

CH3

G:5MeC

G:C

Proper G:C Base Pairs

6MeA

Proper A:T Base Pairs

‐Differentkindsofrestric>onenzymes(blunt/distal)‐Sharedrecogni>onsequences‐Sharedoverhangs

‐Buffercondi>ons‐Storinganddilu>ngyourrestric>onenzymes‐Specificity(poten>alpiValls!)‐Lackofac>vity(hostcells&poten>alpiValls)

On a practical level… Using Restriction Enzymes

Double check you’re using the enzyme you think you are using! 

Figure by Justin Lo

Construction of the Δ5 Plasmid

Anticipating Problems & Pitfalls:

What might go wrong in your experiment?

Incomplete Reactions

Controls: How can you tell if your DNA has actually been cut?

•  AbouttheexperimentsinMod1

– HowdoesDNAdamagecausemuta>ons?

– Howisrecombina>onusedtofixdoublestrandbreaks?

– Overviewoftheexperimentsyouwillbedoing

•  Restric9onEnzymes

–  Basicsrestric>onenzymes

•  An9cipa9ngPoten9alProblems&Pi@alls

– Whatcontrolsareneededandwhy?